Rpl13a

RNA was extracted from sorted cells using the RNeasy Micro Kit (Qiagen, Hilden, Germany), and cDNA was synthesized from 35ng RNA using the Reverse transcription kit (Applied Biosystems, Foster City, CA) according to manufacturer's instructions. Quantitative PCR was performed using the ABI Prism 7700 Sequence Detector (Applied Biosystems). All PCR reactions were performed in triplicates using TaqMan Gene Expression mastermix (Applied Biosystems). For analysis, the genes GAPDH and RPL13A were used as internal control. The following Taqman Probes were purchased from Applied Biosystems: GAPDH (Hs04194366_g1); RPL13A (Hs02786624_g1); CD34 (Hs02576480_m1); GPR56 (Hs00938474_m1); ZBTB46 (Hs01008166_m1); MMRN1 (Hs01113299_m1); CPXM1 (Hs00219709_m1); DPYSLR3 (Hs00181668_m1); SMIM24 (Hs00415400_m1); BEX3 (Hs00276273_s1); DNMT3B (Hs00171876_m1); CDK6 (Hs01026371_m1); SOCS2 (Hs00919620_m1); AKR1C3 (Hs00366267_m1); ARHGAP22 (Hs01098342_m1); LAPTM4B (Hs00363282_m1); EMP1 (Hs00608055_m1); KIAA0125 (Hs00796164_s1); NYNRIN (Hs00394058_m1). The ΔCt values were obtained for every probe after normalization from internal control. A ratio of expression levels of genes in GPR56^hi vs GPR56^lo cells as well as CLL‐1^hi vs CLL‐1^lo cells was calculated.

The adductor muscle group of WT mice was harvested before (pre-treatment = pt) and on different time points (day 1, 3, 7, 14, 28) after hind limb ischemia, snap-frozen, crushed, using mortar and pestle, and homogenized over a Qiashredder (Qiagen). Total RNA was extracted using RNeasy fibrous tissue minikit (Qiagen) according to manufacturer’s instructions and RNA integrity was checked using the nanodrop 1000 Spectrophotometer (nanodrop Technologies) and the 2100 Bioanalyzer (Agilent Technologies).
Total RNA from whole bone marrow and bone marrow derived monocytes was isolated using a standard Trizol-chloroform extraction protocol. RNA concentration, purity and integrity were examined by nanodrop (nanodrop Technologies).
For real-time quantitative PCR, RNA was reverse transcribed using High Capacity RNA-to-cDNA kit (Applied Biosystems). Quantitative PCR was performed on the ABI 7500 Fast system, using commercially available TaqMan gene expression assays for TLR4, CD180 (RP105), SIGIRR, ST2L, CTSS, MMP9, MMP2, PI3K, RAC1, GAPDH and RPL13A (Applied Biosystems). Expression levels of GAPDH and RPL13A were used for normalization.

Total RNA from whole blood cells was isolated by way of the PAXgene Blood RNA kit (Qiagen) according to the manufacturer’s recommendations. Total RNA was obtained from skin samples, cDNA was synthesized, and real-time polymerase chain reactions (quantitative RT-PCR) were performed as previously described (35 (link)). Oligonucleotide sequences used in the RT–qPCR assays are displayed in Supplementary Table 6. The reactions were incubated in the StepOnePlus^® real-time PCR equipment (Applied Biosystems, United States) as described (36 (link)). For each sample, the cycle- threshold (CT) means of the genes of interest (IFNB, IFNAR1, IFI16, TBK1, EIF2AK2, and MX1) were normalized by the CT mean of the reference gene RPL13a (ThermoFisher Scientific, United States). The relative gene expression analysis was performed utilizing the 2^–ΔCT method for each target gene (37 (link)).

RNeasy Mini Kit was used for mRNA isolation according to the manufacturer’s protocol (Qiagen, Hilden, Germany). High-Capacity cDNA Reverse Transcription Kit was used to reverse transcribe RNA to cDNA (Thermo Fisher Scientific, Waltham, MA, USA). The 10 µL PCR reaction included 2 µL RT product, 5 µL TaqMan Gene expression master Mix, 0.5 µL probe mix of the TaqMan (respectively, T-bet, GATA, RORc, FoxP3, GAPDH, TBP, RPL13a—all from Thermo Fisher Scientific, Waltham, MA, USA), and 2.5 µL of water (Genoplast, Poland). Reactions were performed at 50 °C for 2 min, 95 °C for 10 min, followed by 50 cycles at 95 °C for 15 s and 60 °C for 1 min. Samples were analyzed in triplicate using the QuantStudio 5 qRT-PCR machines (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was evaluated using ΔΔCT-method.

Total RNA was isolated from IVD cells using the RNeasy micro kit (# 74004, Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. A NanoDrop™ ND1000 UV-VIS spectrophotometer (Isogen Life Science B.V., Utrecht, Netherlands) was used to measure RNA quantity. cDNA was synthesized from total RNA in a 20 µL reaction volume using the High-Capacity cDNA RT kit (Thermo Fisher), according to the manufacturer’s instructions. qPCR analysis was performed using TaqMan universal master mix II (# 4440043, Thermo Fisher) and probes for human NFATc1 (assay no. Hs00542675_m1, Thermo Fisher) and P2RX7 (assay no. Hs00175721_m1, Thermo Fisher). Experimental reactions were conducted by preincubation (95°C for 10 min) and amplification (95°C for 15 s and 60°C for 60 s) for 40 cycles. RPL13a (assay no. Hs04194366_g1, Thermo Fisher) was used for normalization of mRNA expression. Gene expression was assessed using a CFX96™ PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, United States), and relative gene expression was calculated using the comparative 2^−ΔΔCt method and expressed as fold change. All reactions were performed in triplicate.

Total RNA was isolated with RNeasy Plus Mini Kit (Qiagen), and cDNA was prepared with superscript III First-Strand Synthesis System for RT–PCR kit (Invitrogen). RT–qPCR was done with TaqMan PCR system (Thermo Fisher Scientific) or QuantiTect SYBR Green PCR system (Qiagen). TaqMan probes for Zbtb7b (ThPOK; Mm00784709_s1), Cd40lg (CD40L; Mm00441911_m1), Gata3 (Mm00484683_m1), Eomes (Mm01351985_m1), Prf1 (Perforin; Mm00812512_m1), and Rpl13a (Mm01612986_gH) were from Thermo Fisher Scientific. The primer sequences for SYBR green PCR system were as follows: Runx3d forward (5′- GCGACATGGCTTCCAACAGC-3′) and reverse (5′-CTTAGCGCGCTGTTCTCGC-3′); Rpl13a forward (5′-CGAGGCATGCTGCCCCACAA-3′) and reverse (5′-AGCAGGGACCACCATCCGCT-3′). Samples were analyzed on QuantStudio 6 Flex Real-time PCR System (Applied Biosystems). Gene expression values were normalized to those of Rpl13a expression in the same sample.